title: Cytoplasmic overexpression of CD95L in esophageal adenocarcinoma cells overcomes resistance to CD95-mediated apoptosis.
abstract: BACKGROUND
  The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege.
  
  
  METHODS
  CD95L expression was characterized in immortalized squamous esophagus (HET-1A) and Barrett esophagus (BAR-T) cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control); and KFL cells (+ control). Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways.
  
  
  RESULTS
  Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling.
  
  
  CONCLUSIONS
  CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis.
authors.affiliation: Department of Surgery, Division of Surgical Oncology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Watson
authors.forename: Sanjay
authors.initials: S
authors.lastname: Naran
authors.forename: Xinglu
authors.initials: X
authors.lastname: Zhang
authors.forename: Michael T
authors.initials: MT
authors.lastname: Stang
authors.forename: Pierre E
authors.initials: PE
authors.lastname: Queiroz de Oliveira
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Hughes
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D004938:Esophageal Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D053222:Fas Ligand Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: D053222:Fas Ligand Protein; D012333:RNA, Messenger; D019014:fas Receptor